S6 kinases are components of a pathway essential for cell cycle transit and are effectors of zell-autonomous growth. Three S6 kinases (p70S6K1, p85S6K2, and p54S6K2) derived from two separate genes (S6K1 and S6K2) are currently known, all of which share common regulatory motifs yet are localized to discrete subcellular compartments. The S6 kinases are regulated by complex, multi-site phosphorylation that serves both to disable an auto-inhibitory mechanism and to directly induce enzyme activation. Over-activity or amplification of S6 kinases has been reported in a number of cancers indicating that anti-cancer therapies aimed at inactivation of S6 kinase pathways may prove efficacious. Indeed, compounds which target S6 kinase pathways (i.e., rapamycin and CCI-779) have successfully completed several phase I clinical evaluations as anti-tumor agents. These studies will clarify the regulation and respective functions of individual S6 kinases during mitosis and in cell cycle progression. Also addressed will be the nature and importance of S6 kinase regulation by Pinl during mitosis and throughout the cell cycle. Because S6 kinases are essential for cell cycle progression, and as such, are under evaluation as targets for novel cancer therapies, understanding the function of S6 kinases in cell cycle transit will provide information vital to the design of new anti-cancer strategies.